**3.3 The effect of inoculum concentration**

256 Biogas

for dairy wastes, and 2.3 l H2/l medium for brewery wastes) were observed when 9 and

Fig. 3. Influence of filtration of dairy wastewaters on hydrogen generation.

taking into account that light intensity on sunny day can be higher than 100 klx.

Similar light intensity (8klx) was use by Zhu et al. for tofu wastewaters treated with *Rhodobacter sphaeroides.* Volume of hydrogen obtained for these conditions was 1.5 l H2/l medium (2.8 l H2/l medium when glucose was used) (Zhu, 1999). Nath et al. showed the best results of hydrogen generation when 10 klx was applied (Nath, 2009). Li et al., however, studying the photofermantation of glycerol with *Rhodobacter sphaeroides ZX-5* proved the highest hydrogen production with light intensity not exceeding 5 klx (Li, 2009). Surprisingly, high light intensity was tested by Obeid et al. for photofermentation of lactate medium and *Rhodobacter capsulatus IR3* (up to 50 klx). Highest effectiveness and rate of hydrogen production were obtained when 30-50 klx were used. These tests are essential

Light intensity seems to be an important factor in hydrogen photogenerating process. On one hand increase at light intensity stimulates hydrogen production and biomass growth, on the other hand too high intensity may cause the reduction of nitrogenase activity or even damage of the cells (Asada, 1999, Uvar, 2005). An important parameter which shows the relationship between light intensity, irradiation area, duration of H2 production and total H2 amount is the light conversion efficiency (η, equation 1). It is the ratio of the total energy of the obtained hydrogen to the total energy input of the photobioreactor by solar radiation (Eroğlu, 2007). In our tests η reached the highest value when 9 klx was applied (2.4 % for dairy waste, 1.7 for brewery waste, Table 2) Results in Table 2 show that illumination with

13 klx were applied.

In these series of experiments we tested several concentrations of inoculum introduced to the medium: 5-40 % v/v (0.086 g dry wt/l – 0.48 g dry wt/l) for standard medium and 10% and 30% (0.086 g dry wt/l and 0.36 dry wt/l) in case of medium containing wastes (Fig. 5). The optimum inoculum concentration in all cases turned out to be 30% v/v. Data in Table 3 indicate that the second higher concentration produces more hydrogen, shorter lag phase

Fig. 4. The effect of light intensity on hydrogen production in photofermentation process (30% v/v inoculum, 40% v/v dairy waste, 10% v/v brewery waste)

Hydrogen [l H2/l medium]

0,0

Concentration of dairy waste (% v/v)

5 10 20 40 80 (concentrated)

loss and efficiencies.

0,5

1,0

1,5

5%

10%

20%

30%

40%

2,0

2,5

3,0

3,5

Photofermentative Hydrogen Generation in Presence of Waste Water from Food Industry 259

10%

30%

standard dairy

Fig. 5. The effect of inoculum concentration on hydrogen production

Hmax (l/l medium)

> 0.77 1.58 2.1 3.2 0

> 0.86 1.17 1.4 2.24 0.52

> 0.38 0.4 0.4 0.56 0.67

waste

COD loss (gO2/l medium)

> 1.3 1.8 2.8 4.2 -

> 1.9 2.4 2.8 3.8 2.3

> 1.3 1.5 1.6 2.4 2.8

Dairy waste (COD = 46 g O2/l)

Brewery waste I (COD = 220 g O2/l)

Brewery waste II (COD = 27 g O2/l)

Standard (L-malic acid) 0.2 2.3 1.9 - 1.2

Table 4. The correlation between waste concentration, amount of hydrogen produced, COD

brewery waste

Ysub (l H2/l waste)

> 11.3 13.7 9.4 7.6 -

3.6 2.0 1.0 0.9 0.59

Ysp (l H2/ CODloss)

> 0.6 0.78 0.75 0.76 -

> 0.45 0.49 0.51 0.59 0.23

> 0.29 0.27 0.25 0.23 0.24

10%

30%


and bigger COD loss. Increase to 40% lead to smaller amount of produced H2. This effect seems to be caused by the fact that with the inoculum, apart from biomass, we also introduced metabolites which in high concentrations negatively influence the efficiency of photofermentation (Waligórska, 2006, Koku, 2002).

Table 3. Kinetic parameters of cumulative hydrogen production at different concentration of inoculum

### **3.4 The effect of waste concentration on hydrogen production**

The effect of waste concentration was studied with inoculum concentration of 30% ( 0.36 dry wt/l) and light intensity of 9 klx. The following waste concentration were used: 5, 10, 20, 40, 60% v/v in case of dairy waste, 1, 3, 5, 10, 20% v/v in case of brewery waste I and 5, 10, 20, 40, 80% v/v in case of brewery waste II. The results in Tabl.4 show the maximum hydrogen production of 3.2 l/l medium occurring when 40% of dairy waste was used. When brewery waste with high COD (220 g O2/l) was applied, 2.2 l of H2 per l medium was produced (waste concentration10% v/v). In case of brewery waste with low COD (27 g O2/l) only 0.67 l of H2 per l medium was produced (waste concentration 80 % v/v). If higher concentrations of wastes were applied, the efficiency of hydrogen production was lower, which was caused by and inhibiting concentration of N-NH4 + (40 mg/l for dairy waste and 96 mg/l for brewery waste) (Waligórska, 2009, Melis, 2006). Such concentration of ammonium ions can diminish significantly the overall generation of hydrogen . The presence of ammonium ions as well as N2 causes reduction of nitrogen *via* nitrogenase into gaseous NH3 instead of required hydrogen. The amount of evolved CO2 never exceeded 10 vol. %. Additionally, higher concentrations of wastes caused acidification of medium during the process and darkens the medium, which makes the access of the light into the medium more difficult and negatively impact on hydrogen production. The final pH values presented on fig. 6 show the drop from 7.1 to 5.2 in case of brewery waste and 7.5 to 5.7 in case of dairy waste. This effect is caused mainly by formation of organic acids (lactic and acetic) (Koku, 2002). The higher was the concentration of the waste the higher was the amount of detected acids and lower value of pH. This can be explained by higher ability of transfer of undissociated form of acids towards the cell, followed by dissociation inside the cell, proton release and final inhibition of the process (Van Ginkel, 2005).

and bigger COD loss. Increase to 40% lead to smaller amount of produced H2. This effect seems to be caused by the fact that with the inoculum, apart from biomass, we also introduced metabolites which in high concentrations negatively influence the efficiency of

> λH2 (h)

18.0±7.6 14.5±4.3

11.6±2.9 9.4±2.6

Dairy waste

Brewery waste I

Table 3. Kinetic parameters of cumulative hydrogen production at different concentration of

The effect of waste concentration was studied with inoculum concentration of 30% ( 0.36 dry wt/l) and light intensity of 9 klx. The following waste concentration were used: 5, 10, 20, 40, 60% v/v in case of dairy waste, 1, 3, 5, 10, 20% v/v in case of brewery waste I and 5, 10, 20, 40, 80% v/v in case of brewery waste II. The results in Tabl.4 show the maximum hydrogen production of 3.2 l/l medium occurring when 40% of dairy waste was used. When brewery waste with high COD (220 g O2/l) was applied, 2.2 l of H2 per l medium was produced (waste concentration10% v/v). In case of brewery waste with low COD (27 g O2/l) only 0.67 l of H2 per l medium was produced (waste concentration 80 % v/v). If higher concentrations of wastes were applied, the efficiency of hydrogen production was lower, which was caused

brewery waste) (Waligórska, 2009, Melis, 2006). Such concentration of ammonium ions can diminish significantly the overall generation of hydrogen . The presence of ammonium ions as well as N2 causes reduction of nitrogen *via* nitrogenase into gaseous NH3 instead of required hydrogen. The amount of evolved CO2 never exceeded 10 vol. %. Additionally, higher concentrations of wastes caused acidification of medium during the process and darkens the medium, which makes the access of the light into the medium more difficult and negatively impact on hydrogen production. The final pH values presented on fig. 6 show the drop from 7.1 to 5.2 in case of brewery waste and 7.5 to 5.7 in case of dairy waste. This effect is caused mainly by formation of organic acids (lactic and acetic) (Koku, 2002). The higher was the concentration of the waste the higher was the amount of detected acids and lower value of pH. This can be explained by higher ability of transfer of undissociated form of acids towards the cell, followed by dissociation inside the cell, proton release and

Y (l H2/l waste)

> 5.8 7.6

13.6 19

pH final

> 7.3 6.9

> 6.1 6.2

+ (40 mg/l for dairy waste and 96 mg/l for

COD loss (g O2/l)

> 3.5 4.2

> 3.1 3.8

Biomass (g/l)

> 2.0 2.2

> 2.7 2.6

photofermentation (Waligórska, 2006, Koku, 2002).

Rmax,H2 (l/l/h)

0.057±0.018 0.049±0.007

0.034±0.004 0.061±0.009

**3.4 The effect of waste concentration on hydrogen production** 

Hmax (l/l)

2.52±0.17 3.23±0.21

1.41±0.04 2.24±0.09

by and inhibiting concentration of N-NH4

final inhibition of the process (Van Ginkel, 2005).

Inoculum concentration (g dry wt/l)

> 0.086 0.36

> 0.086 0.36

inoculum


Fig. 5. The effect of inoculum concentration on hydrogen production

Table 4. The correlation between waste concentration, amount of hydrogen produced, COD loss and efficiencies.

production.

Hydrogen [l/l medium]

0

inoculum, 9 klx)

1

2

3

standard

10%brewery waste I 80%brewery waste II 40%dairy waste

Photofermentative Hydrogen Generation in Presence of Waste Water from Food Industry 261

efficiency of hydrogen generation (0.1g/l waste) for low waste concentration (olive mill wastewater 2%) however maximum volume of hydrogen production (0.45 l/l) and highest COD loss (40%) were observed when higher waste concentrations were used (Eroğlu, 2004). Also Mohan et al. studding hydrogen production from vegetable based market waste, obtained good specific efficiency when low waste concentrations were used, however highest COD loss (almost 60%) occurred when higher waste concentrations were introduced to the media (Mohan , 2009). Comparing the above results with the ones obtained for hydrogen generation on standard medium with L-malic acid, it can be seen that total amount of produced hydrogen is by 30% higher when dairy waste in concentration of 40%v/v was used and comparable when brewery waste with high COD was used (Table 4, Fig 7). Different papers published so far have proved that organic substrates such as glucose, sucrose, malic acid have been more effective than the waste containing media (Yetis2000, Zhu, 1999, Basak, 2009). However based on our results we can state that wastes studied in this paper represent an effective nutrient for photobiological hydrogen

time [h]

0 20 40 60 80 100

Fig. 7. The effect of optimum waste concentration on hydrogen production (30% v/v

Fig. 6. Influence of food wastewater concentration on pH, biomass increase and COD loss.

With the rising concentration of wastes we observed higher COD loss, biomass increase and increase of specific efficiency (Table 4, fig.6). With further increase of waste concentration COD loss and specific efficiency were lower. However substrate efficiency decreases with higher waste concentration. Similar results showed Eroglu et al. obtaining the best substrate

pH biomass

p H biom ass

5% 10% 20% 40%

increase

b) dairy waste Fig. 6. Influence of food wastewater concentration on pH, biomass increase and COD loss.

With the rising concentration of wastes we observed higher COD loss, biomass increase and increase of specific efficiency (Table 4, fig.6). With further increase of waste concentration COD loss and specific efficiency were lower. However substrate efficiency decreases with higher waste concentration. Similar results showed Eroglu et al. obtaining the best substrate

60%

5% 10%

1% 3% 5%

increase

a) brewery waste (Seifert, 2010)

10% 20%

COD loss

COD loss

60%

20%

40%

10%

20%

**a**

**b**

1% 3% 5%

pH, biomass [g dry wt/l], COD [g O2/l]

0

pH, biomass [g dry wt/l], COD [g O2/l]

0

2

4

6

8

2

4

6

8

1%

5% 10%

20% 40%

60%

3% 5%

10%

20%

efficiency of hydrogen generation (0.1g/l waste) for low waste concentration (olive mill wastewater 2%) however maximum volume of hydrogen production (0.45 l/l) and highest COD loss (40%) were observed when higher waste concentrations were used (Eroğlu, 2004). Also Mohan et al. studding hydrogen production from vegetable based market waste, obtained good specific efficiency when low waste concentrations were used, however highest COD loss (almost 60%) occurred when higher waste concentrations were introduced to the media (Mohan , 2009). Comparing the above results with the ones obtained for hydrogen generation on standard medium with L-malic acid, it can be seen that total amount of produced hydrogen is by 30% higher when dairy waste in concentration of 40%v/v was used and comparable when brewery waste with high COD was used (Table 4, Fig 7). Different papers published so far have proved that organic substrates such as glucose, sucrose, malic acid have been more effective than the waste containing media (Yetis2000, Zhu, 1999, Basak, 2009). However based on our results we can state that wastes studied in this paper represent an effective nutrient for photobiological hydrogen production.

Fig. 7. The effect of optimum waste concentration on hydrogen production (30% v/v inoculum, 9 klx)

Photofermentative Hydrogen Generation in Presence of Waste Water from Food Industry 263

Brewery waste

standard 2.3±0.2 0.047±0.004 2.7±1.8

The presented results shows that the waste studied in this paper represent a vary good substrate in photophermentation by *Rhodobacter sphaeroides.* Light intensity of 9 klx and inoculum concentration of 0.36 g dry wt/l (30% v/v) were used as the most effective (high light conversion efficiency and short duration of the process). The studied wastes has to be treated with high temperature (20 min in 120oC). This pretreatment significantly increases H2 production. The optimum concentrations of wastes were estimated: 40% v/v for dairy waste and 10% v/v for brewery waste with high COD. These wastes represent the effective (comparable with L-malic acid) nutrient for hydrogen production. Higher wastes concentrations inhibit the process as it initiate fermentation which starts to compete with

affect the process. Brewery waste with low COD shows low efficiencies and needs to be concentrated to supply sufficient concentration of organic compounds. An application of untreated dairy wastewater containing suspensions in efficient hydrogen generation process can be performed only at controlled acidity (pH = 7.0). Kinetic measurements proved that the rate of hydrogen generation drops with concentration of the waste and prolongs the lag

These studies were supported by Polish Ministry of Science and Higher Education (grant

Apha, AWWA, WEF. Standard methods for the examination of water and wastewater. 10th

ed. Washington, DC: American Public Health Association; 1995.

0.77±0.03 1.58±0.11 2.10±0.06 3.23±0.21

0.86±0.02 1.17±0.05 1.40±0.05 2.24±0.09 0.52±0.02

Table 6. Kinetic parameters of cumulative hydrogen production for different initial

(% v/v) Hmax (l/l) Rmax (l/l/h) λH2 (h) Dairy waste

> 0.08±0.05 0.058±0.019 0.055±0.021 0.049±0.007

> 0.046±0.007 0.045±0.009 0.042±0.008 0.061±0.009 0.040±0.015

6.5±3.1 7.3±6.2 10.0±4.8 14.5±4.3

8.0±1.4 6.1±2.7 6.1±2.1 9.4±2.6 18.7±2.2

+ concentration, which also negatively

Concentration of waste

concentration of food waste

hydrogen production and additionally increases NH4

**4. Conclusions** 

phase.

**5. Acknowledgements** 

no: N N204 185440).

**6. References** 

#### **3.5 The influence of pH correction on hydrogen production**

The untreated "raw" dairy wastewater with low value of pH (4.27) was completely nonactive in hydrogen generation by microbiological method. However, we assumed that the same wastewater under controlled pH can generate hydrogen similarly as a sterilized one. Therefore, in order to achieve similar conditions like in bioreactor operating under controlled pH we performed our batch tests in small photoreactors (capacity of 60 ml with working capacity of 30 ml) correcting pH with 0.5M solution of NaOH every 12 h. Medium containing non-sterilized dairy wastewater with concentration of 40 v/v % was inoculated with bacteria at two different concentrations: 0.086g dry wt/l (10 vol.%) or 0.36 g dry wt/l (30 vol.%). Data presented in table 5 indicate that stabilization of the system at pH close to 7 allows for hydrogen generation even from the untreated dairy wastewater. Application of inoculums with concentration at the 0.36 g dry wt/l level generates 3.6 l H2/l. The four-fold dilution of microorganisms reduces the volume of hydrogen to 2.6 l H2/l. Although the starting time was relatively long (about 20 h) savings which could arise from the application of untreated waste can be significant. Performing the same experiment with brewery waste II ( concentration 40 v/v % ) the yield of the generated hydrogen has not been improved. In this case the value of pH rapidly grew to 7.5- 7.9 in the first two days. However , it can not be excluded that in the system with controlled pH this yield could be much higher. Preliminary experiments performed under such conditions confirm this assumption.


\* expressed in g dry wt/l

\*\* biomass increase

Table 5. Kinetic parameters of cumulative hydrogen production for non-treated 40 % dairy wastewater, with correction of pH for different concentration of inoculums (Seifert, 2010).

The results presented in this section suggest that hydrogen generation can be effectively performed under solar radiation in photobioreactor operating under continuous conditions.

#### **3.6 Kinetic of hydrogen generation**

The results of kinetic considerations based on modified Gompertz equation (Eq. 4) are shown in table 6. Independently from the kind of food waste (in the active of concentration) it was observed that the increase of the volume of generated hydrogen, small drops in reaction rate and prolongation of the lag phase.

These results showed that higher substrate yield increases the reaction rate. Moreover, these values are well correlated with the lag phase in systems with higher concentration of wastes are caused probably by longer adaptation of microorganisms to the bed.


Table 6. Kinetic parameters of cumulative hydrogen production for different initial concentration of food waste
